Cloning and characterization of Pseudomonas putida genes encoding the phosphate-specific transport system

The pstSCAB genes of Pseudomonas putida PRS2000, encoding the phosphate (P- i)-specific transport (Pst) system, were cloned. The pstS gene of Pseudomon as aeruginosa PAO1, of which the pstCAB genes had been cloned previously, w as also cloned (Nikata, T. et al., Mel. Gen. Genet., 250, 692-698, 1996). T he predicted translation products of the P. putida pstSCAB genes showed 83, 75, 78 and 88% amino acid identity with their P. aeruginosa counterparts. Two well-conserved Pho box sequences were found in the region upstream of t he pstS gene (15/18 base identity with the consensus Pho box sequence) and in the intercistronic region between the pstS and pstC genes (11/18 base id entity) of P. putida PRS2000. To investigate the functions of PstSCAB, the pstSC genes were inactivated by inserting a kanamycin resistance gene casse tte into the chromosome of P. putida PRS2000. The resultant mutant, designa ted PNT1, failed to take up P-32(i) even under conditions of P-i limitation . Strain PNT1 was also constitutive for alkaline phosphatase synthesis, as well as chemotaxis toward P-i, indicating that the Pst system is involved i n the negative regulation of the pho regulon in P. putida. Although overexp ression of the pstSCAB genes in P. putida PRS2000 resulted in decreased cel l growth, this recombinant strain could remove P-i at a rate similar to tha t seen with the control strain.